Changes in antioxidant activity of fresh marine macroalgae from the Canary Islands during air-drying process

Algal Research 66 (2022) 102798

Available online 31 July 2022

2211-9264/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Changes in antioxidant activity of fresh marine macroalgae from the Canary Islands during air-drying process

Marcos Adri ´ an Ruiz-Medina

^*

, Marta Sans ´ on, Agueda María Gonz ´ ´ alez-Rodríguez

Departamento de Bot´anica, Ecología y Fisiología Vegetal, Universidad de La Laguna, 38200 Santa Cruz de Tenerife, Canary Islands, Spain

A R T I C L E I N F O Keywords:

Bioactive compounds DPPH

Ferrous iron-chelating Flavonoids Phenolic compounds Phytoconstituent

A B S T R A C T

Marine macroalgae develop structurally distinct molecules that have been long recognized as an important source of natural products, e.g., bioactive compounds and antioxidants, such as carotenoids, amino acids, pro- teins, lipids, vitamins and polyphenols, with promising properties as anticoagulant, anti-proliferative and anti- microbial. In this study we analyzed the antioxidant activity of 24 fresh methanolic extracts from marine macroalgae from the Canary Islands. Additionally, the effects of air-drying process in the antioxidant activity were also evaluated. Plant material was collected in three locations on the island of Tenerife. Total antioxidant activity methods included DPPH-free radical scavenging activity and ferrous iron chelating activity. Phytocon- stituents evaluated were total carotenoids, proline, phenols, flavonoids and condensed tannins content. Our results indicated that from fresh material, species Cladophora liebetruthii, Dasycladus vermicularis and Dictyopteris polypodioides presented the highest scavenging activity, supported by high correlation with phenolics and fla- vonoids content, however, in air-dry extracts, Anadyomene saldanhae and D. polypodioides showed the highest antioxidant potential, correlated with a high phenolic compounds content. Asteronema breviarticulatum and D. polypodioides presented a high content of carotenoids in fresh and air-dry state. The highest ferrous iron chelating activity (>60 %) was recorded in extracts from fresh material in Grateloupia imbricata and Lophocladia trichoclados. However, only L. trichoclados was able to maintain this high activity after air-drying supported by a high proline content. A. breviarticulatum was the species that showed the highest condensed tannins content both in fresh and dried extracts. The study revealed that fresh extracts of D. vermicularis, D. polypodioides and L. trichoclados possess promising properties as raw materials to obtain biologically active substances in alimentary and pharmaceutical industry. Besides, these properties were maintained after the air-drying period in the last two species, which makes them species of great interest to be used under drying preservation methods.

1. Introduction

Oxidative stress is an unavoidable consequence of life in an oxygen- rich atmosphere. The human body maintains a constant oxide-reduction balance, preserving the balance between the production of substances that induce oxidative stress and the antioxidant defense systems [1]. The loss in this oxide-reduction balance leads to a state of oxidative stress that is characterized by an increase in the levels of free radicals (FRs) and reactive species, which is not compensated by the antioxidant de- fense systems, causing damage and cell death [2]. Reactive species include oxygen species (ROS) and there are many pathological processes reasonably attributable to ROS attack, some of the most significant are:

aging and cell death, cancer, mutations and heart, muscular, pulmonary

and liver diseases, among others [3–5]. Fortunately, the negative effects of oxidative stress may be mitigated by antioxidants [4]. Marine or- ganisms, including macroalgae, have attracted the attention of many researchers as a source of bioactive compounds and antioxidants due to their properties, the diversity of their molecules, and their new chemical structures that are complex and difficult to synthesize chemically [6].

Many studies have already demonstrated the promising properties of marine macroalgae extracts, being an excellent source of bioactive compounds such as carotenoids, amino acids, proteins, lipids, pigments, vitamins and polyphenols, among other substances [7,8]. Carotenoids are largely distributed in lipoproteins and membranes, usually together with vitamin E [9]. Main antioxidant capabilities of carotenoids include pro-vitamin A activity, immunological, endocrine and metabolic

Abbreviations: dry wt, dry weight; TPC, total phenolic content; TFC, total flavonoid content; CTC, condensed tannin content; FIC, ferrous iron-chelating.

* Corresponding author.

E-mail addresses: alu0101051642@ull.edu.es (M.A. Ruiz-Medina), msanson@ull.edu.es (M. Sans´on), aglerod@ull.edu.es (A.M. Gonz´ ´alez-Rodríguez).

Contents lists available at ScienceDirect

Algal Research

journal homepage: www.elsevier.com/locate/algal

https://doi.org/10.1016/j.algal.2022.102798

Received 2 February 2022; Received in revised form 16 July 2022; Accepted 17 July 2022

activities [10,11]. In addition, the amino acid proline, an essential osmolyte in the management of water stress in plants, has pharmaco- logical characteristics that are still unknown [12]. It is currently known that proline is involved in collagen synthesis, as well as the preservation of joints and the prevention of arthritis, the improvement of tissue and mucous membrane healing, the prevention or improvement of diseases like ulcers, cardiovascular disorders and the maintenance of skin, bone and muscle tissue [12,13].

Even though in nature there are multiple compounds with antioxi- dant activity, phenolic compounds are probably the most studied. They have also been shown to have potential as antidiabetics, anticancer, anti-inflammatory, antihypertensive, anticoagulant, antimicrobial and antiallergic, which gives them great value in the alimentary and phar- maceutical industry [14]. Furthermore, within this large group are fla- vonoids, which are a particular type of polyphenols present in plants that are also well known [15]. The growing interest in flavonoids is due to the appreciation of their broad pharmacological activity. Due to this fact, protective effects have been described in pathologies such as can- cer, heart diseases, viral infections, stomach and duodenal ulcer and inflammations [16]. Flavonoids include condensed tannins, which are known by their usefulness in the treatment of allergies, cancers, car- diovascular disorders, and platelet aggregation [17].

Macroalgae are commonly classified into three higher taxa, brown (Class Phaeophyceae), red (Phylum Rhodophyta) and green (Phylum Chlorophyta) [18]. According to Guiry & Guiry [19] about 2092 species of brown algae, 6984 of green algae and 7470 of red algae are worldwide currently recognized. All these organisms, like other photosynthetic plants, are exposed to a combination of light and oxygen that leads to the formation of FRs and other strong oxidizing agents, besides, some of them live in complex habitats exposed to extreme conditions (high salinity, low or high temperature, high pressure, low availability of nutrients, and low or high exposure to sunlight) that further increase oxidative stress [20]. In addition, the absence of structural and photo- dynamic induced damage suggests that algae possess mechanisms for a rapid adaptation, producing secondary metabolites, which protect them against effects of oxidative stress in unfavorable periods [21,22].

An immense algal biodiversity develops on the Canary Islands, currently >600 species are recognized [23,24]. Many of these species have a ubiquitous distribution and therefore have already been studied to find compounds with biological activity, for example, ethyl acetate extract of Ulva rigida presented a high concentration of total phenols while U. lactuca presented also a high total phenolic content [25,26].

Other species that have shown high antioxidant, anti-inflammatory and antiproliferative activity have been those of the Cystoseira s.l. [27].

However, many other species are distributed regionally and their morphological and physiological characteristics (small size, morphology, growth habits) make their collection difficult and, there- fore, have led to few or incomplete studies on their biological potential.

Fresh algae collected or cultivated from the sea are usually dried before being used in any nutritional evaluation or industrial processing as they originally consist of 70–90 % water [28]. This large amount of moisture irretrievably leads to rapid microbial decomposition and the destruction of biologically active substances [29]. The best procedure to preserve the flavor, texture, and nutritional value of macroalgae after harvesting is freezing because considerably reduces the physical and biochemical changes involved in molecules degradation, as well as the development and reproduction of spoilage microbes [30,31]. However, when large warehouses with freezers are not available, air-drying could be a preservation method. Drying is the traditional algae preservation procedure and could be an important factor affecting the nutritional value of species either through chemical modifications or direct losses of the nutrients [32]. Exposure to the atmosphere places a stress on marine macroalgae when their tissues dry out. Previous studies have described that prolonged period of drying induce oxidative stress through ROS overproduction, alterations in cell membranes and protein denaturation, disruption of the life cycle and finally cell death [33,34]. However, there

are many physiological mechanisms that can be activated to attenuate oxidative stress, for example, increasing the antioxidant response or accumulating compatible solutes or osmolytes that help avoid osmotic stress [35,36].

Even though marine macroalgae are known to contain a wide variety of secondary compounds, comprehensive chemical analysis, and the identification of bioactive components in many species, as well as the effects of drying procedure on the antioxidant properties, remain un- known [37,38]. Therefore, the objectives of this study were (1) to evaluate the antioxidant activity of methanolic extracts from 24 species of marine macroalgae from the island of Tenerife (Canary Islands) and (2) to analyze the effects of air-drying as a processing and storage method on this activity. Total antioxidant activity methods included DPPH-free radical scavenging activity and ferrous iron chelating activ- ity. Phytoconstituents evaluated were total carotenoids, proline, phe- nols, flavonoids and condensed tannins content.

2. Material and methods

2.1. Study area and macroalgae sampling

In Canary Islands, subtropical waters are characterized by a quasi- permanent thermocline caused by the strong surface heating throughout the year. The thermocline is only eroded from January to March, allowing cold nutrient-rich waters to enter the euphotic zone and thereby increasing primary productivity during the so-called “late winter bloom” [39]. Besides, the islands are located at the outer edge of the intertropical convergence zone, so they get a quantity of Saharan dust, which can fertilize the water column with phosphorus and iron (1–3 nM), among other trace metals [40,41]. In Tenerife, sea surface temperature annually ranges from 17 ^◦C (February) to 24 ^◦C (September) [42]. In this study, 24 species of macroalgae (3 Phaeo- phyceae, 7 Chlorophyta and 14 Rhodophyta) belonging to 10 orders were collected between October–November 2021 from three sites located on the north of Tenerife (Table 1): 15 species in Punta del Hi- dalgo (28^◦34^′15.57^′′ N, 16^◦19.59^′83^′′ O), 4 in Punta Brava (28^◦24^′45.08^′′N, 16^◦33.57^′68^′′O) and 5 in El Penitente (28^◦25^′07.30^′′

N, 16^◦32^′55.08^′′O) (Fig. 1).

2.2. Plant material processing

Samples, 10 g per individual (3 individuals per species) were washed to remove sand, salts and cleaned off epiphytes. To analyze antioxidant activity in fresh, half of the cleaned material were immediately frozen and stored at − 80 ^◦C. The other half was subjected to a drying treatment, which consisted in air-dry on filter paper during one week in a darkness chamber with constant temperature (19 ±2 ^◦C) and air humidity con- ditions (80 ±5 %), measured with a thermo-hygrometer (EMS33, EMS, Brno, CZ). After one week, the samples were frozen and stored at

− 80 ^◦C. The frozen material was lyophilized, ground until obtaining a fine powder and finally stored at − 20 ^◦C until later assays.

2.3. Preparation of algal extracts for antioxidant activity and phenolic compounds determination

To execute radical DPPH scavenging activity and ferrous iron chelating activity assays, in addition to determining the content of total phenolics, flavonoids and condensed tannins, the same extraction was performed following Xu and Chang [43] protocol with slight modifica- tions. 0.015 g of dry powder was suspended in 2 mL of solvent [80:20 methanol/water (80 % methanol)] and vortexed vigorously during 30 s.

Subsequently, it was kept stirring for 1,5 h at 400 rpm and then allowed to stand over the night. Next day the supernatant was collected in a test tube that was reserved. The pellet was resuspended again in another 2 mL of solvent and vortexed vigorously during 30 s. Finally, both su- pernatants were unified, filtered with a syringe through a 0.45 μm

Millipore filter, and stored at − 20 ^◦C in the dark until further analysis.

The extractions were performed in triplicate.

2.4. DPPH free radical scavenging activity assay

DPPH-free radical scavenging capacity of leaf extracts was evaluated according to the method of Chen and Ho [44] with slight modifications.

Briefly, 0.2 mL of the extract was added to 3.8 mL of 0.25 mM ethanolic DPPH solution. The mixture was vigorously stirred for 1 min and allowed to stand at room temperature in the dark for 30 min. Next, the absorbance of the sample plus DPPH solution (Asample) was measured using the UV–visible spectrophotometer (UV-160 A, Shimadzu) at 517 nm against an ethanol blank. A negative control (Acontrol) was taken after adding DPPH solution to 0.2 mL of the solvent (80 % methanol). The percentage of discoloration by DPPH of the sample was calculated

according to the equation:

Percentage of discoloration=[ 1− (

Asample

/Acontrol

) ]×100

The FR scavenging activity of the extracts was expressed as equiva- lent to that of Trolox. Results were calculated and expressed as milli- grams of Trolox equivalents (TE) per gram of dry weight using the Trolox calibration curve. The linearity range of the calibration curve was 50 to 350 μg/mL (r =0.99).

2.5. Ferrous ion-chelating activity assay

The ferrous iron-chelating (FIC) activity assay was adapted from a method described by Gülçin et al. [45] with Chew et al. [46] modifi- cations. A reaction mixture was prepared by mixing 0.1 mM ferrous sulphate, 0.25 mM FerroZine and macroalgae extract in 1:1:1 propor- tion. The reaction mixture was allowed to stand for 10 min before the absorbance measurements were taken at 562 nm using a UV–visible spectrophotometer (UV-160 A, Shimadzu). The ferrous ion-chelating property of extracts was calculated as a percentage using the following formula:

Chelating activity(%) =[(

Acontrol− Asample

)/Acontrol

]×100

where Acontrol and Asample are the absorbance of the control and extract, respectively. The negative control contained 80 % methanol, ferrous sulphate and FerroZine in 1:1:1 proportion. EDTA was used as positive control at same the concentration of extracts.

2.6. Determination of total phenolic content

The total phenolic content (TPC) was determined by a Folin- Ciocalteu assay [47,48] using gallic acid (GA) as the standard. The mixture of the sample solution (50 μL), distilled water (1.5 mL), 250 μL of 2 N Folin-Ciocalteu's reagents solution, and 7 % sodium carbonate (750 μL) was vortexed and incubated for 8 min at room temperature.

Then, a dose of 950 μL of distilled water was added. The mixture was allowed to stand for 2 h at room temperature. Finally, the absorbance was measured immediately against the blank (distilled water) at 765 nm using a UV–visible spectrophotometer (UV-160 A, Shimadzu) and the results were calculated and expressed as milligrams of GA equivalents (mg GAE/g dry wt) using the GA calibration curve (10–400 μg/mL; r = 0.99).

2.7. Determination of total flavonoid content

The total flavonoid content (TFC) was determined by a previously described colorimetric method with slight modifications [49]. Briefly, to 250 μL of extract or standard solution of (+)-catechin was added 1.25 mL of distilled water in a test tube, followed by the addition of 75 μL of a 5 % sodium nitrite solution. After 6 min rest, 150 μL of a 10 % aluminum chloride solution were added and allowed to stand for another 5 min before adding 0.5 mL of 1 M sodium hydroxide. The mixture was made up to 2.5 mL with distilled water and mixed well. The absorbance was measured immediately against the blank (the same mixture without the sample) at 510 nm using a UV–visible spectrophotometer (UV-160 A, Shimadzu). Results were calculated and expressed as micrograms of (+)-catechin equivalents (mg CAE/g dry wt) using the (+)-catechin calibration curve. The linearity range of the calibration curve was 10 to 400 μg/mL (r =0.99).

2.8. Determination of condensed tannin content

Analysis of condensed tannin content (CTC) was carried out ac- cording to the method of Broadhurst and Jones [50]. To 100 μL of the sample extract, 2 mL of a 4 % vanillin methanol solution and 1 mL of concentrated hydrochloric acid (12 M) were added. The mixture was Table 1

Taxonomic classification and collection sites of the 24 marine macroalgal species.

Name of the species and authority Taxonomy Collection site Anadyomene saldanhae A.B.Joly & E.C.

Oliveira Ch, Cladophorales,

Anadyomenaceae PH

Asteronema breviarticulatum (J.Agardh)

Ouriques & Bouzon Ph, Scytothamnales, Asteronemataceae PE Canistrocarpus cervicornis (Kützing) De

Paula & De Clerck Ph, Dictyotales,

Dictyotaceae PH

Caulerpa webbiana Montagne Ch, Bryopsidales,

Caulerpaceae PH

Cladophora albida (Nees) Kutzing Ch, Cladophorales,

Cladophoraceae PH

Cladophora liebetruthii Grunow Ch, Cladophorales,

Cladophoraceae PH

Codium intertextum Collins & Hervey Ch, Bryopsidales,

Codiaceae PH

Dasycladus vermicularis (Scopoli)

Krasser Ch, Dasycladales,

Dasycladaceae PH

Dictyopteris polypodioides (A.P.De

Candolle) J.V.Lamouroux Ph, Dictyotales,

Dictyotaceae PH

Galaxaura rugosa (J.Ellis & Solander) J.

V.Lamouroux Rh, Nemaliales,

Galaxauraceae PH

Grateloupia imbricata Holmes Rh, Halymeniales,

Halymeniaceae PB

Heterodasya mucronata (Harvey) M.J.

Wynne Rh, Ceramiales,

Rhodomelaceae PH

Hypnea spinella (C.Agardh) Kützing Rh, Gigartinales,

Cystocloniaceae PH

Hypnea sp. Rh, Gigartinales,

Cystocloniaceae PB

Jania pedunculata var. adhaerens (J.V.

Lamouroux) A.S.Harvey, Woelkerling

& Reviers

Rh, Corallinales,

Corallinaceae PE

Jania virgata (Zanardini) Montagne Rh, Corallinales,

Corallinaceae PE

Laurencia dendroidea J.Agardh Rh, Ceramiales,

Rhodomelaceae PE

Laurencia pyramidalis Bory ex Kützing Rh, Ceramiales,

Rhodomelaceae PB

Lophocladia trichoclados (C.Agardh) F.

Schmitz Rh, Ceramiales,

Rhodomelaceae PH

Palisada perforata (Bory) K.W.Nam Rh, Ceramiales,

Rhodomelaceae PH

Rytiphlaea tinctoria (Clemente) C.

Agardh Rh, Ceramiales,

Rhodomelaceae PH

Spyridia filamentosa (Wulfen) Harvey Rh, Ceramiales,

Callithamniaceae PB Valonia utricularis (Roth) C.Agardh Ch, Cladophorales,

Valoniaceae PE

Wrangelia argus (Montagne) Montagne Rh, Ceramiales,

Wrangeliaceae PH

The taxonomic abbreviations Rh, Ph and Ch represent Phylum Rhodophyta, Class Phaeophyceae and Phylum Chlorophyta, respectively. The locality ab- breviations PH, PB and PE represent Punta del Hidalgo, Punta Brava y El Pen- itente, respectively.

vortexed during 5 s and left to stand for 15 min, and the absorption was measured using a UV–visible spectrophotometer (UV-160 A, Shimadzu) at 500 nm against 80 % methanol as a blank. CTC was calculated and expressed as mg catechin equivalents (mg of CAE/g dry wt) using the calibration curve of (+)-catechin. Linearity range of the calibration curve was 10 to 400 μg/mL (r =0.99).

2.9. Determination of total carotenoids content

For carotenoids determination, 0.01 g of dry powder were extracted with 2 mL 100 % acetone saturated with calcium carbonate in order to avoid acid traces that modify the pigment composition. They were centrifuged for 10 min at 4 ^◦C and syringe-filtered through 0.45 μm Millipore filter. Total carotenoids were quantified with a double beam spectrophotometer (UV-160 A, Shimadzu) following the method pro- posed by Lichtenthaler [51], for which it is necessary to calculate the concentration of chlorophylls a and b. The equations for calculating total carotenoids are shown below:

Total carotenoids= [(1000×A470) − (1.9×Ca) − (63.14×Cb) ]/214 Chlorophylla(Ca) = (11.24×A661.5) − (2.04×A644.5)

Chlorophyllb(Cb) = (20.13×A644.5) − (4.19×A661.5)

Results are expressed as mg/g dry weight (dry wt). The extraction was conducted in triplicate.

2.10. Determination of total proline content

The colorimetric analysis of proline was carried out according to Bates et al. [52] based on proline's reaction with ninhydrin. Dry powder (0.03 g per sample) was extracted with 2 mL 3 % sulfosalicylic acid and centrifuged 5 min at 1400 rpm. For determinations, 1:1:1 solution of supernatant, ninhydrin acid and glacial acetic acid was incubated at 100 ^◦C for 1 h. The content was poured into a tube with 2 mL of toluene to extract the chromophore, then vortexed 1 min and centrifuged 5 min at 1000 rpm. Finally, the chromophore was placed in the upper layer and its absorbance at 520 nm was determined in a UV–visible spectro- photometer (UV-160 A, Shimadzu). The extraction was conducted in triplicate. The method was calibrated for each determination with standard L-proline solutions within the detection range of the method (1–300 μM; r =0.99).

2.11. Statistical analysis

Statistical analyses were performed using SPSS 25.0 software pack- age (SPSS, Inc., Chicago, IL, USA). Normality and homoscedasticity of

samples distribution was assessed using Shapiro Wilk and Levene's test, respectively. Differences between fresh and dried macroalgae extracts were analyzed by Student t-test. Duncan's multiple range test was car- ried out to test any significant differences among species, while Pearson correlation test was conducted to determine the correlation coefficient (r) among quantitative variables. Significant levels were defined using p

≤0.05.

3. Results

3.1. Total antioxidant activity

Antioxidant activity of marine macroalgae extracts is summarized in Table 2. In eight species, the radical scavenging activity increased significantly after air-drying, for example in A. saldanhae, H. spinella and J. pedunculata var. adhaerens, whose dried samples (23.12 ±0.64, 16.43

±0.73 and 15.16 ±0.49 mg TE/g dry wt, respectively) were up to nine times higher than the fresh samples (8.12 ±0.30, 1.86 ±0.08 and 1.82

± 0.04 mg TE/g dry wt, respectively). However, two species (C. liebetruthii and D. vermicularis) showed an opposite effect, with a radical scavenging activity in fresh extract significantly higher (29.15 ± 0.75 and 58.71 ±0.81 mg TE/g dry wt) than in dry extract (10.37 ± 0.87 and 21.53 ±0.72 mg TE/g dry wt, respectively).

Significant differences were found among species. In extracts made from fresh material, D. vermicularis presented the highest activity (58.71 mg TE/g dry wt), which translated into a discoloration percentage of 47

%, significantly higher than the rest of the species. It was followed by C. liebetruthii and D. polypodioides which presented a discoloration around 23 %. In the dry extracts, the highest values were recorded in D. polypodioides (29.92 ±0.55 mg TE/g dry wt) and A. saldanhae (23.12

±0.64 mg TE/g dry wt), significantly different from each other but higher than the rest of the species.

The FIC activity of the analyzed extracts is shown in Fig. 2. FIC ac- tivity decreased significantly after the drying period in 50 % of the macroalgae studied. This was revealed in C. cervicornis and G. imbricata whose activity in fresh (59.22 ±0.45 and 73.81 ±0.67 %, respectively) was significantly higher than in dry (31.96 ±0.72 and 30.56 ±0.68 %, respectively). However, in 5 species this activity was favored after water loss, for example in W. argus, whose values were 55.11 ±0.55 % and 29.22 ±0.45 % in dry and fresh, respectively. After a week of air-drying, only J. virgata lost all the FIC activity. Significant differences were also found between species, the highest values compared to the control (EDTA, 92.70 ±0.74 %) were recorded in G. imbricata in fresh (73.81 %) and in L. trichoclados in fresh (67.89 %) and dry (74.48 %). In general, all species presented high FIC activity values, being in 5 species >50 % (C. cervicornis, D. vermicularis, A. breviarticulatum, L. trichoclados and G. imbricata).

Fig. 1. Geographical location of the Canary Islands and collection sites in Tenerife Island: Punta Brava, El Penitente and Punta del Hidalgo.

3.2. Total phenols and phenolic compounds content

The TPC of the fresh and dry algae material is shown in Table 3. After the drying period, no significant differences were found between fresh and dried material in the studied species. Only two species of green algae showed a decrease in the TPC after the loss of water, C. liebetruthii and D. vermicularis, from fresh values of 33.14 ±1.55 and 41.59 ±1.75 mg GAE/g dry wt, to 12.83 ±0.83 and 17.97 ±0.50 mg GAE/g dry wt after drying, respectively. In fresh samples, the highest significant TPC was reached by D. vermicularis followed by A. breviarticulatum, C. liebetruthii and D. polypodioides, which showed a similar high content, around 33 mg GAE/g dry wt. From dry material, the highest values observed were presented by D. polypodioides (31.43 ± 0.90 mg GAE/g dry wt) and A. breviarticulatum (29.65 ± 1.31 mg GAE/g dry wt), which were

significantly higher than the rest of the species. Finally, A. saldanhae and R. tinctoria also presented a high content, about 21 mg GAE/g dry wt.

Additionally, the TFC decreased after the drying period in half of the studied species (Table 3), being the only exception R. tinctoria that showed a value of 5.42 ±0.26 mg CAE/g dry wt in fresh condition and 9.10 ±0.24 mg CAE/g dry wt in dry condition. The decrease in flavo- noid content after the drying period was significant in 54 % of the species, being in some cases up to 5 times lower, such as in P. perforata, whose TFC was 4.15 ±0.17 mg CAE/g dry wt in fresh state and 0.74 ± 0.12 mg CAE/g dry wt after drying. Among species, D. vermicularis (16.23 ±0.18 mg CAE/g dry wt) exhibited the highest value in the extracts obtained from fresh material, which was significantly higher than the rest of the species. The following were A. saldanhae and D. polypodioides, with values close to 10 mg CAE/g dry wt. After drying, the highest values corresponded to A. saldanhae (9.42 ±0.28 mg CAE/g dry wt) and R. tinctoria, which were significantly higher than the rest of the species.

Within the flavonoids, CTC showed greater sensitivity to drying time (Table 3), since in most species their concentration decreased signifi- cantly after a week of air drying, even zero values were recorded, as for example in S. filamentosa and G. imbricata, which initially had values of 3.56 and 1.26 mg CAE/g dry wt, respectively. Among species, the highest concentration of condensed tannins was presented by A. breviarticulatum (20.96 ±0.71 mg CAE/g dry wt), significantly higher than the other species. C. cervicornis, D. polypodioides and C. webbiana showed the next highest values from fresh material, with a concentra- tion around 12 mg CAE/g dry wt. The same trend was observed from dried material.

3.3. Total carotenoids and proline content

Significant differences in the total carotenoids content were found between fresh and dry samples in all species (Fig. 3), being higher in fresh than in dry material. In some species carotenoids decreased significantly, up to five or ten times the content in the extracts from fresh macroalgae, which was the case of the Hypnea spp. and S. filamentosa. In the rest of the species, the total carotenoid values tended to decrease up to 50 % after drying, with respect to the values in fresh. Among the species, the highest values were presented in fresh material of A. breviarticulatum (1.09 ±0.00 mg/g dry wt), H. spinella (1.15 ±0.00 mg/g dry wt) and D. polypodioides (1.19 ±0.01 mg/g dry wt), which differed significantly with the rest of the species. Regarding the dry material, the total content of carotenoids was highly variable among species. A. breviarticulatum showed the highest value after air-drying (0.94 mg/g dry wt), in addition to other species that also maintained good levels such as A. saldanhae and V. utricularis, (0.4 and 0.57 mg/g dry wt, respectively). Therefore, these values were significantly higher than other species that presented higher values when fresh but that after air-drying were more affected, for example, Hypnea spp. and H. mucronata.

As observed in Fig. 4, the dried samples presented a higher quantity of total proline content, with some exceptions such as in L. dendroidea, whose total content of this amino acid from fresh material was 31.38 ± 0.48 μmol/g dry wt, significantly higher than the material after drying (8.54 ±0.14 μmol/g dry wt). In other species, such as R. tinctoria, no significant differences were observed between the dry and fresh states.

In the comparison among species, the highest values in both fresh and dry material were obtained in L. trichoclados (105.62 ±0.54 and 127.38

±0.29 μmol/g dry wt, respectively) that were significantly higher than the rest of the species (up to 100 times higher in some cases).

3.4. Correlation analyses between phytochemicals and total antioxidant activity

Regardless of the material (fresh or dried), correlation analyses be- tween phytochemicals content and total antioxidant activities among all Table 2

DPPH-free radical scavenging activity from 24 marine macroalgae extracts (fresh and after a week of air-drying). Data are expressed as mean ±standard error (n =3).

Plant name DPPH (mg TE/g dry wt) DPPH (discoloration %) Fresh

algae Dried algae Fresh

algae Dried algae Heterodasya mucronata 1.79 ±

0.00^A 2.21 ±

0.15^AB 0.00 ±

0.00^A 0.35 ± 0.13^AB Lophocladia

trichoclados 1.82 ±

0.04^A 7.43 ±

0.95*^C 0.03 ±

0.03^A 4.70 ± 0.79*^C Jania pedunculata var.

adhaerens 1.82 ±

0.04^A 15.16 ±

0.49*^E 0.03 ±

0.03^A 11.14 ± 0.41*^E

Wrangelia argus 1.82 ±

0.04^A 3.64 ±

0.71^AB 0.03 ±

0.03^A 1.55 ± 0.59^AB

Hypnea spinella 1.86 ±

0.08^A 16.43 ±

0.73*^E 0.06 ±

0.06^A 12.21 ± 0.61*^E Valonia utricularis 1.86 ±

0.08^A 1.90 ±

0.07^AB 0.06 ±

0.06^A 0.10 ± 0.06^AB

Cladophora albida 1.90 ±

0.12^A 1.90 ±

0.12^AB 0.10 ±

0.10^A 0.10 ± 0.10^AB Codium intertextum 1.90 ±

0.12^A 1.86 ±

0.04^AB 0.10 ±

0.10^A 0.06 ± 0.03^AB Spyridia filamentosa 1.90 ±

0.12^A 6.89 ±

0.61*^C 0.10 ±

0.10^A 4.25 ± 0.51*^C

Galaxaura rugosa 1.94 ±

0.10^A 1.86 ±

0.04^AB 0.13 ±

0.09^A 0.06 ± 0.03^AB Laurencia pyramidalis 1.94 ±

0.10^A 3.52 ±

0.57^AB 0.13 ±

0.09^A 1.45 ± 0.48^AB

Palisada perforata 1.94 ±

0.10^A 1.82 ±

0.04^A 0.13 ±

0.09^A 0.03 ± 0.03^A

Hypnea sp. 1.98 ±

0.10^A 2.87 ±

0.20*^AB 0.16 ±

0.09^A 0.90 ± 0.17*^AB Grateloupia imbricata 2.09 ±

0.17^A 3.06 ±

0.35^AB 0.26 ±

0.14^A 1.06 ± 0.30^AB Canistrocarpus

cervicornis 2.13 ±

0.18^A 5.88 ±

0.76*^C 0.29 ±

0.15^A 3.41 ± 0.63*^C

Caulerpa webbiana 2.13 ±

0.20^A 2.37 ±

0.13^AB 0.29 ±

0.17^A 0.48 ± 0.11^AB Laurencia dendroidea 2.52 ±

0.22^A 3.10 ±

0.27^AB 0.61 ±

0.18^A 1.10 ± 0.23^AB

Jania virgata 2.91 ±

0.41^A 2.02 ±

0.13^AB 0.93 ±

0.34^A 0.19 ± 0.11^AB Rytiphlaea tinctoria 4.45 ±

0.40^B 18.83 ±

0.85*^F 2.22 ±

0.33^B 14.20 ± 0.71*^F Anadyomene saldanhae 8.12 ±

0.30^C 23.12 ±

0.64*^H 5.28 ±

0.25^C 17.78 ± 0.53*^H Asteronema

breviarticulatum 17.59 ±

0.67^D 20.22 ±

0.57*^FG 13.17 ±

0.56^D 15.36 ± 0.48*^FG Dictyopteris

polypodioides 28.07 ±

0.4^E 29.92 ±

0.55^I 21.90 ±

0.40^E 23.45 ± 0.45^I Cladophora liebetruthii 29.15 ±

0.75*^F 10.37 ±

0.87^D 22.80 ±

0.62*^F 7.15 ± 0.73^D Dasycladus vermicularis 58.71 ±

0.81*^G 21.53 ±

0.72^G 47.44 ±

0.68*^G 16.46 ± 0.60^G Asterisks indicate significant differences between fresh and dry extracts in each species and different capital letters indicate significant differences among different species in each state: fresh or air-dried (p ≤0.05).

macroalgae samples were performed (Table 4). Among the analyzes carried out from fresh material, except for the total proline content and CTC, the rest of the phytoconstituents were linearly correlated signifi- cantly (p ≤0.01) with the total antioxidant activity measured with the DPPH assay. By comparing the correlation coefficients (r) between the variables, it was found that antioxidant activity was mainly enhanced by TPC (r =0.88) and TFC (r =0.85). On the other hand, FIC activity had a significant correlation (p ≤0.01) with total proline content (r =0.37)

and CTC (r =0.26), although in this case it was lower. However, when the material was dried, DPPH radical scavenging activity was strongly and positively correlated (p ≤0.01) with TPC (r =0.84), TFC (r =0.62) and CTC (r =0.50), while FIC activity in fresh was similar to dry ma- terial. In general, carotenoids were significantly correlated with TPC and phenolic compounds. Proline content was not significantly correlated with any phytoconstituent.

Fig. 2. Ferrous ion-chelating (FIC) activities of methanol extracts from 24 species of algae fresh and after a week of air drying. EDTA (ethylenediaminetetraacetic acid) was used as positive control. Data are expressed as mean ±standard error (n =3). Asterisks indicate significant differences between fresh and dry extracts in each species and different capital letters indicate significant differences among different species in each state: fresh or air-dried (p ≤0.05).

Table 3

Total phenolic, flavonoid and condensed tannin content from 24 marine macroalgae extracts (fresh and after a week of air-drying). Data are expressed as mean ± standard error (n =3).

Plant name TPC (mg GAE/g dry wt) TFC (mg CAE/g dry wt) CTC (mg CAE/g dry wt)

Fresh algae Dried algae Fresh algae Dried algae Fresh algae Dried algae

Jania pedunculata var. adhaerens 2.54 ±0.61^A 3.17 ±0.72^AB 1.52 ±0.07^A 1.16 ±0.24^A 1.71 ±0.52^ABC 0.22 ±0.13^AB

Valonia utricularis 2.67 ±0.29^A 3.17 ±0.66^AB 4.17 ±0.31*^CD 2.54 ±0.28^BC 3.41 ±0.52*^DEF 0.15 ±0.15^A

Codium intertextum 2.73 ±0.50^A 3.43 ±0.77^ABC 4.70 ±0.37*^DE 2.73 ±0.13^BC 0.44 ±0.26^AB 0.15 ±0.07^A

Spyridia filamentosa 2.73 ±0.44^A 3.49 ±0.61^ABC 2.98 ±0.28^B 2.14 ±0.15^B 3.56 ±0.51*^DEF 0.00 ±0.00^A

Galaxaura rugosa 3.24 ±0.50^AB 3.43 ±0.83^ABC 1.93 ±0.20*^A 1.04 ±0.15^A 2.96 ±0.33*^CDE 0.07 ±0.07^A

Jania virgata 3.43 ±0.40^AB 3.68 ±0.44^ABC 1.54 ±0.15*^A 0.68 ±0.09^A 0.00 ±0.00^A 0.00 ±0.00^A

Grateloupia imbricata 4.19 ±0.72^ABC 2.79 ±0.35^A 1.74 ±0.11^A 1.44 ±0.11^A 1.26 ±0.39*^ABC 0.00 ±0.00^A

Wrangelia argus 4.25 ±1.00^ABC 4.25 ±0.88^ABCD 1.71 ±0.19^A 1.12 ±0.12^A 7.78 ±0.77*^H 1.93 ±0.71^CD

Hypnea sp. 4.32 ±0.46^ABC 5.02 ±0.39^ABCD 2.22 ±0.10*^A 1.25 ±0.12^A 5.04 ±0.45*^FG 0.00 ±0.00^A

Cladophora albida 5.27 ±0.50^ABC 5.90 ±0.83^CDEF 3.05 ±0.17^B 3.04 ±0.24^CD 2.89 ±0.71^CDE 0.96 ±0.45^ABC

Hypnea spinella 5.52 ±0.40^BC 5.59 ±0.35^BCDE 1.97 ±0.13*^A 1.14 ±0.13^A 3.41 ±0.52*^DEF 0.67 ±0.13^AB

Laurencia pyramidalis 5.78 ±0.61^BCD 6.60 ±0.61^DEFG 4.85 ±0.28*^EF 3.56 ±0.24^D 3.18 ±0.58*^DE 0.22 ±0.13^AB

Lophocladia trichoclados 6.29 ±0.66^CDE 8.89 ±0.72^G 3.94 ±0.36^CD 2.58 ±0.33^BC 2.52 ±0.45^CDE 3.33 ±0.64^EF

Heterodasya mucronata 6.41 ±0.61^CDE 4.38 ±0.88^ABCD 1.50 ±0.14*^A 0.78 ±0.19^A 2.15 ±0.45^BCD 1.19 ±0.32^ABC

Canistrocarpus cervicornis 6.48 ±0.61^CDE 5.65 ±0.78^BCDE 6.71 ±0.20^H 5.42 ±0.44^E 12.52 ±0.71*^I 5.48 ±0.77^G

Laurencia dendroidea 8.32 ±0.78^DE 7.05 ±0.83^EFG 6.07 ±0.13^GH 5.86 ±0.28^EF 4.15 ±0.85*^EFG 0.89 ±0.34^ABC

Palisada perforata 8.70 ±0.92^EF 7.81 ±0.61^FG 4.15 ±0.17*^CD 0.74 ±0.12^A 2.37 ±0.39*^CDE 0.37 ±0.20^AB

Caulerpa webbiana 10.92 ±0.66^F 7.68 ±0.94^FG 5.97 ±0.22^G 5.59 ±0.33^EF 11.11 ±0.77*^I 4.07 ±0.45^F

Rytiphlaea tinctoria 18.92 ±0.72^G 20.76 ±0.99^J 5.42 ±0.26^FG 9.10 ±0.24*^H 5.71 ±0.58^G 3.48 ±0.52^EF

Anadyomene saldanhae 21.21 ±1.27^G 20.70 ±0.99^J 9.67 ±0.38^J 9.42 ±0.28^H 2.30 ±0.32*^CDE 0.15 ±0.15^A

Cladophora liebetruthii 33.14 ±1.55*^H 12.83 ±0.83^H 8.47 ±0.20*^I 6.31 ±0.32^FG 3.56 ±0.51*^DEF 1.48 ±0.27^BC

Asteronema breviarticulatum 33.59 ±1.28^H 29.65 ±1.31^K 6.69 ±0.15^H 6.60 ±0.17^G 20.96 ±0.71*^J 15.48 ±0.61^I

Dictyopteris polypodioides 33.97 ±0.88^H 31.43 ±0.90^K 9.67 ±0.24*^J 5.84 ±0.26^EF 11.19 ±0.96^I 7.70 ±0.77^H

Dasycladus vermicularis 41.59 ±1.75*^I 17.97 ±0.50^I 16.23 ±0.18*^K 6.96 ±0.37^G 2.00 ±0.34^BCD 2.74 ±0.27^DE

Asterisks indicate significant differences between fresh and dry extracts in each species and different capital letters indicate significant differences among different species in each state: fresh or air-dried (p ≤0.05).

4. Discussion

An in-depth screening has been performed for determination of the antioxidant properties of 24 marine macroalgae extracts, emphasizing the effect of air-drying process (under controlled conditions) as a pro- cessing and storage method in these properties. It has been found that, regardless of their phylogenetic group or genus, the studied species exhibited a variable antioxidant activity. In some species this activity decreased after the air-drying experiment. Therefore, the role of pro- cessing and storage must be considered for preservation of the antioxi- dant activity in macroalgae.

In this study the highest potential scavenging activity was presented by D. vermicularis, whose percentage of discoloration was above other species of macroalgae such as Chondrus and Porphyra spp. [21]. In

addition, other studied macroalgae, such as A. breviarticulatum and D. polypodioides, also showed a high antioxidant capacity. Similar data was obtained by Karaki et al. [53], where D. polypodioides showed high antioxidant and anticoagulant capacity from isolated polysaccharides.

Moreover, those species possess the ability to maintain the antioxidant activity after air-drying, which make them candidate species for com- mercial use in contrast to other species of edible macroalgae which maintain a lower total antioxidant activity after air-drying, e.g., Petal- onia binghamiae [54] as well as Fucus sp., which antioxidant activity by DPPH decreased by 96 % [21].

On the other hand, all methanolic extracts analyzed in this study presented FIC activity that mean that the transition metal ion Fe²⁺ possess the ability to move single electrons by virtue of which it can allow the formation and propagation of many radical reactions [55].

Fig. 3. Total carotenoids content in methanol extracts from 24 species of algae fresh and after a week of air drying. Data are expressed as mean ±standard error (n

=3). Asterisks indicate significant differences between fresh and dry extracts in each species and different capital letters indicate significant differences among different species in each state: fresh or air-dried (p ≤0.05).

Fig. 4.Total proline content in methanol extracts from 24 species of algae fresh and after a week of air drying. Data are expressed as mean ±standard error (n =3).

Asterisks indicate significant differences between fresh and dry extracts in each species and different capital letters indicate significant differences among different species in each state: fresh or air-dried (p ≤0.05).

Among analyzed species, only J. virgata lost its activity after air-drying, which did not occur in J. pedunculata var. adhaerens, what it is indicative that each species has its own bioactive molecules that allow them to live and adapt to their environment. In our study, a high capacity as a chelating agent (>60 %) was observed in fresh extracts of G. imbricata and L. trichoclados (close to the EDTA control). This capacity was maintained after the air-drying process in L. trichoclados. Today we know that this chemical substance, as well as its sodium salts, have numerous applications in industry, cosmetic products, laboratory and in medicine, where it is used for the treatment of mercury and lead poisoning through the chelation therapy [56]. Thus, these properties make L. trichoclados a species of high interest.

Antioxidant activity of phenolic compounds is correlated to their chemical structures. In general, FRs scavenging and antioxidant activity of phenolics mainly depends on the number and position of hydrogen- donating hydroxyl groups on the aromatic ring of the phenolic mole- cules [57]. In our study we found two species (C. liebetruthii and D. vermicularis) with a high content of total phenolic compounds, how- ever, after air-drying this content decreased significantly as happened in Hormosira banksii and other edible marine macroalgae [21,58]. Excep- tionally, A. breviarticulatum and D. polypodioides presented a high phenolic content that was maintained after drying, being even in D. polypodioides up to 4 times higher than fresh extract from other spe- cies of the genus, for example D. divaricata [59]. In our study, D. vermicularis presented the highest concentration of flavonoids in fresh material, which was positively correlated with the highest antioxidant activity (DPPH assay). Nevertheless, although other studies have shown the ability of this species to produce high concentrations of polyphenolic compounds in response to high radiation and low salinity [60,61], we have demostrated that the opposite effect occurs after prolonged air- drying, having a significant decrease in flavonoids content. Otherwise, tannins like phlorotannins are mostly found in marine algae and they are phloroglucinol oligomers produced in brown algae and have tannin activities. Possibly the activity of phlorotannins in macroalgae is related to the presence of condensed tannins [62], and therefore in this study the highest concentration of tannins was found in brown algae A. breviarticulatum, C. cervicornis and D. polypodioides, which is consis- tent with that found in other species of brown algae such as Sargassum ringgoldianum [63] and Macrocystis integrifolia [64].

Among the various defense strategies to reduce oxidative stress, ca- rotenoids are most likely involved in the scavenging of two of the reactive oxygen species, singlet molecular oxygen (¹O2), but mostly peroxyl radicals [65]. In our study, the range of variation of total ca- rotenoids from fresh material varied between 0.15 and 1.2 mg/g dry wt,

while in other studies on edible macroalgae did not detect the presence of carotenoids e.g., Eisenia bicyclis, Sargassum horneri or Cystoseira hakodatensis [66] nor even in microalgae, e.g., Isochrysis galbana [67], However, among all the antioxidants studied, carotenoids were the most affected by air-drying and might be attributed to both down-regulation of biosynthesis and consumption by scavenging superoxide anion [68].

After a prolonged air-drying period, these molecules lose their activity because of their poor stability and high sensitivity to oxidation and degradation, which accordingly limit their applications as health- beneficial components [69].

Proline is a compatible osmolyte, whose concentration increases in plants exposed to air-drying stress. The exogenous application of proline has been seen to significantly improve the content of rosmarinic acid and endogenous proline, which may be indicative of the stimulation of the pentose phosphate pathway. The earliest precursor of rosmarinic acid is erythrose-4-P, which is one of the end products of the pentose phosphate pathway. In consequence, high proline synthesis induces the pentose phosphate pathway and the synthesis of shikimate, rosmarinic acid and other phenolic compounds [70,71]. Therefore, proline content of edible plants may be accepted as a measure of its antioxidant capacity [12,72]. Nevertheless, in our study we did not find a correlation be- tween proline content and phenolic compounds, so further studies may be required to evaluate the role of proline as an antioxidant in marine macroalgae. Among red macroalgae, L. dendroidea, R. tinctoria and L. trichoclados presented the highest proline values, both from fresh and dry material extracts. To our knowledge, the proline concentration of L. trichoclados (127 μmol/g dry wt) is one of the highest recorded in algae, up to 100 times more than in other edible species such as those of the genus Chlorella (C. salina and C. vulgaris). This suggests a great po- tential for endogenous proline synthesis in this species that could be applied in the pharmaceutical industry due to the benefits that this amino acid offers to human health [12].

High correlation detected among total antioxidant activity and phenolic compounds was found in our study, what have been also shown in other studies [73,74]. However, we noted that correlations of the antioxidant activities from different assay methods were also affected by the state of the material (fresh or dried). The findings from the corre- lation analyses indicated that extracts from different material had different degrees of contributions to the overall antioxidant activities of macroalgae. Therefore, for antioxidant studies, the state of the algae material must be carefully selected in each case.

For the first time, a comprehensive study was carried out on phyto- constituents with antioxidant activity in 24 marine macroalgae located in the Canary Islands, comparing the fresh state of the material after Table 4

Correlations between phytoconstituents and total antioxidant activity of 24 marine macroalgae extracts (fresh and air-dry after one week air-drying).

Correlation coefficients (r) DPPH FIC activity Total carotenoids Total proline TPC TFC CTC

Fresh algae (N =72)

DPPH – 0.26* 0.32* − 0.14 0.88** 0.85** 0.14

FIC activity – −0.03 0.37* 0.20 0.21 0.26*

Total carotenoids – − 0.19 0.43* 0.34* 0.60**

Total proline – −0.10 −0.05 − 0.10

TPC – 0.85** 0.40*

TFC – 0.23

CTC –

Dry algae (N =72)

DPPH – 0.19 0.44* 0.01 0.84** 0.62** 0.50**

FIC activity – 0.18 0.58** 0.23 0.18 0.46*

Total carotenoids – − 0.14 0.62** 0.55** 0.72**

Total proline – 0.03 −0.01 0.08

TPC – 0.73** 0.72**

TFC – 0.44*

CTC –

* Correlation is significant at 0.05 level (2-tailed).

** Correlation is significant at 0.01 level (2-tailed).